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Chromatin loop extrusion by Structural Maintenance of Chromosome (SMC) complexes is thought to underlie intermediate-scale chromatin organization inside cells. Motivated by a number of experiments suggesting that nucleosomes may block loop extrusion by SMCs, such as cohesin and condensin complexes, we introduce and characterize theoretically a composite loop extrusion factor (composite LEF) model. In addition to an SMC complex that creates a chromatin loop by encircling two threads of DNA, this model includes a remodeling complex that relocates or removes nucleosomes as it progresses along the chromatin, and nucleosomes that block SMC translocation along the DNA. Loop extrusion is enabled by SMC motion along nucleosome-free DNA, created in the wake of the remodeling complex, while nucleosome re-binding behind the SMC acts as a ratchet, holding the SMC close to the remodeling complex. We show that, for a wide range of parameter values, this collection of factors constitutes a composite LEF that extrudes loops with a velocity, comparable to the velocity of remodeling complex translocation on chromatin in the absence of SMC, and much faster than loop extrusion by an isolated SMC that is blocked by nucleosomes., Comment: 12 pages, 7 figures

SummaryAll cells must fold their genomes, including bacterial cells where the chromosome is compacted into a domain-organized meshwork called nucleoid. Polymer conformation depends highly on the quality of the solvent. Yet, the solvent quality for the DNA polymer inside cells remains unexplored. Here, we developed a method to assess this fundamental physicochemical property in live bacteria. By determining the DNA concentration and apparent average mesh size of the nucleoid, we provide evidence that the cytoplasm is a poor solvent for the chromosome inEscherichia coli. Monte Carlo simulations showed that such a poor solvent compacts the chromosome and promotes spontaneous formation of chromosomal domains connected by lower-density DNA regions. Cryo-electron tomography and fluorescence microscopy revealed that the (poly)ribosome density within the nucleoid is spatially heterogenous and correlates negatively with DNA density. These findings have broad implications to our understanding of chromosome folding and intracellular organization.

SummaryLiquid-liquid phase separation (LLPS) has emerged as a major driver of cellular organization. However, it remains unexplored whether the mechanical properties of LLPS domains are functionally important. The heterochromatin protein HP1-α(and the orthologous Swi6 inS. pombe) is capable of LLPSin vitroand promotes formation of LLPS heterochromatin domainsin vivo. Here, we demonstrate that LLPS of Swi6 contributes to the emergent mechanical properties of nuclei. Using nuclear fluctuation analysis in live cells and force spectroscopy of isolated nuclei, we find that disrupting histone H3K9 methylation or depleting Swi6 compromises nuclear stiffness, while heterochromatin spreading through loss of the H3K9 demethylase, Epe1, increases nuclear stiffness. Leveraging a separation-of-function allele, we demonstrate that phase separation of Swi6—but not its histone binding or dimerization—is essential for Swi6’s mechanical role. These findings demonstrate that altering chromatin state has mechanical consequences and highlights that phase-separated domains can do mechanical work.

All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.

All cells must fold their genomes, including bacterial cells where the chromosome is compacted into a domain-organized meshwork called nucleoid. Polymer conformation depends highly on the quality of the solvent. Yet, the solvent quality for the DNA polymer inside cells remains unexplored. Here, we developed a method to assess this fundamental physicochemical property in live bacteria. By determining the DNA concentration and apparent average mesh size of the nucleoid, we provide evidence that the cytoplasm is a poor solvent for the chromosome in Escherichia coli. Monte Carlo simulations showed that such a poor solvent compacts the chromosome and promotes spontaneous formation of chromosomal domains connected by lower-density DNA regions. Cryo-electron tomography and fluorescence microscopy revealed that the (poly)ribosome density within the nucleoid is spatially heterogenous and correlates negatively with DNA density. These findings have broad implications to our understanding of chromosome folding and intracellular organization.

Several recent experiments, including our own in the fission yeast, S. pombe, have characterized the motions of gene loci within living nuclei by measuring the locus position over time, then proceeding to obtain the statistical properties of this motion. To address the question of whether a population of single particle tracks, obtained from many different cells, corresponds to a single mode of diffusion, we derive theoretical equations describing the probability distribution of the displacement covariance, assuming the displacement is a zero-mean multivariate Gaussian random variable. We also determine the corresponding theoretical means, variances, and third central moments. Bolstering the theory is good agreement between its predictions and the results obtained for various simulated and measured data sets, including simulated particle trajectories of simple and anomalous diffusion, and the measured trajectories of an optically-trapped bead in water, and in a viscoelastic solution. We also show that, for sufficiently long tracks, each covariance distribution in these examples is well-described by a skew-normal distribution with mean, variance, and skewness given by theory. For experimental S. pombe gene locus data, however, we find that the first two covariance distributions are wider than predicted, although the third and subsequent covariances are well-described by theory. This suggests that the origin of the theory-experiment discrepancy is associated with localization noise, which influences only the first two covariances. Thus, we hypothesize that the discrepancy is caused by locus-to-locus heterogeneity in the localization noise. Further simulations reveal excess covariance widths can be largely recreated on the basis of heterogeneous noise. We conclude that the motion of gene loci in fission yeast is consistent with a single mode of diffusion., Comment: Main text 19 pages, 22 figures (28 pages total)

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.

Spatial organization is a hallmark of all living systems. Even bacteria, the smallest forms of cellular life, display defined shapes and complex internal organization, showcasing a highly structured genome, cytoskeletal filaments, localized scaffolding structures, dynamic spatial patterns, active transport, and occasionally, intracellular organelles. Spatial order is required for faithful and efficient cellular replication and offers a powerful means for the development of unique biological properties. Here, we discuss organizational features of bacterial cells and highlight how bacteria have evolved diverse spatial mechanisms to overcome challenges cells face as self-replicating entities.

Summary (Abstract)Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed Genetically Encoded Multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can tune the effective diffusion coefficient of macromolecules ≥15 nm in diameter more than 2-fold without any discernable effect on the motion of molecules ≥5 nm. These mTORCI-dependent changes in crowding and rheology affect phase-separation bothin vitroandin vivo.Together, these results establish a role for mTORCI in controlling both the biophysical properties of the cytoplasm and the phase-separation of biopolymers.

Spatial ordering of macromolecular components inside cells is important for cellular physiology and replication. In bacteria, ParA/B systems are known to generate various intracellular patterns that underlie the transport and partitioning of low-copy-number cargos such as plasmids. ParA/B systems consist of ParA, an ATPase that dimerizes and binds DNA upon ATP binding, and ParB, a protein that binds the cargo and stimulates ParA ATPase activity. Inside cells, ParA is asymmetrically distributed, forming a propagating wave that is followed by the ParB-rich cargo. These correlated dynamics lead to cargo oscillation or equidistant spacing over the nucleoid depending on whether the cargo is in single or multiple copies. Currently, there is no model that explains how these different spatial patterns arise and relate to each other. Here, we test a simple DNA-relay model that has no imposed asymmetry and that only considers the ParA/ParB biochemistry and the known fluctuating and elastic dynamics of chromosomal loci. Stochastic simulations with experimentally derived parameters demonstrate that this model is sufficient to reproduce the signature patterns of ParA/B systems: the propagating ParA gradient correlated with the cargo dynamics, the single-cargo oscillatory motion, and the multicargo equidistant patterning. Stochasticity of ATP hydrolysis breaks the initial symmetry in ParA distribution, resulting in imbalance of elastic force acting on the cargo. Our results may apply beyond ParA/B systems as they reveal how a minimal system of two players, one binding to DNA and the other modulating this binding, can transform directionally random DNA fluctuations into directed motion and intracellular patterning.

In bacteria, ParABS systems mediate intracellular transport of various cargos, including chromosomal regions in Caulobacter crescentus . Transport of the ParB/ parS partition complex requires the DNA-binding activity of ParA, which transiently tethers the partition complex during translocation. In C. crescentus , the directionality of the transport is set up by a gradient of ParA whose concentration gradually increases from one end of the cell (old pole) to the other (new pole). Importantly, this ParA gradient is already observed before DNA replication and segregation are initiated when the partition complex is anchored at the old pole. How such micron-scale ParA pattern is established and maintained before the initiation of chromosome segregation has not been experimentally established. Although the stimulation of ParA ATPase activity by the localized ParB/ parS partition complex is thought to be involved, this activity alone cannot quantitatively describe the ParA pattern observed inside cells. Instead, our experimental and theoretical study shows that the missing key component for achieving the experimentally observed steady-state ParA patterning is the slow mobility of ParA dimers ( D ∼10 −3 μ m 2 /s) due to intermittent DNA binding. Our model recapitulates the entire steady-state ParA distribution observed experimentally, including the shape of the gradient as well as ParA accumulation at the location of the partition complex. Stochastic simulations suggest that cell-to-cell variability in ParA pattern is due to the low ParA copy number in C. crescentus cells. The model also accounts for an apparent exclusion of ParA from regions with small spacing between partition complexes observed in filamentous cells. Collectively, our work demonstrates that in addition to its function in mediating transport, the conserved DNA-binding property of ParA has a critical function before DNA segregation by setting up a ParA pattern required for transport directionality.

Eukaryotic cells spatially organize mRNA processes such as translation and mRNA decay. Much less is clear in bacterial cells where the spatial distribution of mature mRNA remains ambiguous. Using a sensitive method based on quantitative fluorescence in situ hybridization, we show here that in Caulobacter crescentus and Escherichia coli, chromosomally expressed mRNAs largely display limited dispersion from their site of transcription during their lifetime. We estimate apparent diffusion coefficients at least two orders of magnitude lower than expected for freely diffusing mRNA, and provide evidence in C. crescentus that this mRNA localization restricts ribosomal mobility. Furthermore, C. crescentus RNase E appears associated with the DNA independently of its mRNA substrates. Collectively, our findings show that bacteria can spatially organize translation and, potentially, mRNA decay by using the chromosome layout as a template. This chromosome-centric organization has important implications for cellular physiology and for our understanding of gene expression in bacteria.

After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity.

Mossbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding Mossbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps

The use of a scanning flow cytometer (SFC) to study the evolution of monomers, dimers and higher multimers of latex particles at the initial stage of the immunoagglutination is described. The SFC can measure the light-scattering pattern (indicatrix) of an individual particle over an angular range of 10-60 deg. A comparison of the experimentally measured and theoretically calculated indicatrices allows one to discriminate different types of latex particles (i.e. monomers, dimers, etc.) and, therefore, to study the evolution of immunoagglutination process. Validity of the approach was verified by simultaneous measurements of light-scattering patterns and fluorescence from individual polymer particles. Immunoagglutination was initiated by mixing bovine serum albumin (BSA)-covered latex particles (of 1.8 um in diameter) with anti-BSA IgG. The analysis of experimental data was performed on the basis of a mathematical model of diffusion-limited immunoagglutination aggregation with a steric factor. The steric factor was determined by the size and the number of binding sites on the surface of a latex particle. The obtained data are in good agreement with the proposed mathematical modeling., 12 pages, 6 figures

The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria. DOI: http://dx.doi.org/10.7554/eLife.02758.001

A statistical approach is presented to model the kinetics of cell distribution in the process of ligand–receptor binding on cell surfaces. The approach takes into account the variation of the amount of receptors on cells assuming the homogeneity of monovalent binding sites and ligand molecules. The analytical expressions for the kinetics of cell distribution have been derived in the reaction-limited approximation. In order to demonstrate the applicability of the mathematical model, the kinetics of binding the rabbit, anti-mouse IgG with Ig-receptors of the murine hybridoma cells has been measured. Anti-mouse IgG was labeled with fluorescein isothiocyanate (FITC). The kinetics of cell distribution on ligand–receptor complexes was observed during the reaction process by real-time measuring of the fluorescence and light-scattering traces of individual cells with the scanning flow cytometer. The experimental data were fitted by the mathematical model in order to obtain the binding rate constant and the initial cell distribution on the amount of receptors.

SummaryThe physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.

Self-replication is an essential attribute of life but the molecular-level mechanisms involved are not well understood. Cellular self-replication requires not only duplicating all cellular components and doubling volume and membrane area, but also replicating cellular geometry. A whole-cell modeling framework is presented in which an assumed reaction network determines both concentration changes of cellular components and cell geometry. Cell shape is calculated by minimizing membrane-bending energy. Using this framework, simultaneous doubling of volume, surface area, and all components was found to be insufficient to provide mid-cell "pinching" of the parental cell to form two daughter cells. This prompted the design of a minimal protocell that includes a growing shell, a cell-cycle engine, and a contractile ring to enforce cytokinesis. Kinetic parameters were found such that the system exhibited periodic behavior with fundamental aspects of self-replication. This involved simultaneous doubling of all cellular components during a cell cycle, doubling cell volume and membrane area, achieving periodic changes in surface/volume ratio, and forming daughter cells that were geometrically equivalent to each other and to the "newborn" parental cell. The results presented here impact the design of laboratory protocells and the development of a modular strategy for constructing a comprehensive in silico whole-cell model.

Cytokinesis in prokaryotes involves the assembly of a polymeric ring composed of FtsZ protein monomeric units. The Z ring forms at the division plane and is attached to the membrane. After assembly, it maintains a stable yet dynamic steady state. Once induced, the ring contracts and the membrane constricts. In this work, we present a computational deterministic biochemical model exhibiting this behavior. The model is based on biochemical features of FtsZ known from in vitro studies, and it quantitatively reproduces relevant in vitro data. An essential part of the model is a consideration of interfacial reactions involving the cytosol volume, where monomeric FtsZ is dispersed, and the membrane surface in the cell's mid-zone where the ring is assembled. This approach allows the same chemical model to simulate either in vitro or in vivo conditions by adjusting only two geometrical parameters. The model includes minimal reactions, components, and assumptions, yet is able to reproduce sought-after in vivo behavior, including the rapid assembly of the ring via FtsZ-polymerization, the formation of a dynamic steady state in which GTP hydrolysis leads to the exchange of monomeric subunits between cytoplasm and the ring, and finally the induced contraction of the ring. The model gives a quantitative estimate for coupling between the rate of GTP hydrolysis and of FtsZ subunit turnover between the assembled ring and the cytoplasmic pool as observed. Membrane constriction is chemically driven by the strong tendency of GTP-bound FtsZ to self-assembly. The model suggests a possible mechanism of membrane contraction without a motor protein. The portion of the free energy of GTP hydrolysis released in cyclization is indirectly used in this energetically unfavorable process. The model provides a limit to the mechanistic complexity required to mimic ring behavior, and it highlights the importance of parallel in vitro and in vivo modeling., Author Summary The division of a cell into two daughter cells is an essential characteristic of living systems. This process requires the assembly of a contractile ring attached to the membrane at the site of division. At the appropriate point of the cell cycle, the ring constricts and pulls the membrane with it, pinching the cell at the division plane and forming daughter cells. In bacteria, the ring is a polymer of the protein FtsZ. The biochemical features of the isolated FtsZ protein have been studied intensively, but how these features are related to the function of the ring in living cells is not clear. In this work, we mathematically modeled the dynamics of FtsZ ring assembly, steady-state maintenance, and contraction both inside and outside of a cellular environment. The model is extremely simple and includes only experimentally known features of the FtsZ protein. The model exhibited behavior associated with the FtsZ ring in living cells, indicating that no additional mechanistic features are needed to explain this behavior. Our systems biology approach has been successful in relating molecular-level chemical processes with cellular-level behavior. A similar approach might be used to develop an analogous model for the more complex contractile rings found in eukaryotic (e.g., human) cells.

Acetyl-CoA synthases/carbon monoxide dehydrogenases are found in homoacetogenic bacteria, methanogenic archaea, and CO-utilizing hydrogenogenic bacteria.1–4 These O2-sensitive bifunctional enzymes allow such organisms to grow chemo-autotrophically on simple inorganic compounds. The enzyme from the homoacetogen Moorella thermoacetica (ACS/CODH) has been studied most extensively.5,6 The β subunits of this 310 kDa α2β2 tetramer catalyze the reversible reduction of CO2 to CO, while the α subunits catalyze the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from a corrinoid-iron sulfur protein (CoFeSP). At a buffered pH, this is represented by reaction 1. CO+CoA+CH3−Co3+FeSP⇄CH3−C(O)−CoA+Co1+FeSPKACS [1] The active-site for this reaction, called the A-cluster, consists of an [Fe4S4] cubane bridged via a cysteine residue to a Ni ion called proximal Nip. This Ni is also bridged (via 2 other cysteines) to a second Ni ion (distal Nid); thus, Nip is coordinated to 3 bridging thiolates. Nid has an N2S2 square-planar environment including coordination to two amide nitrogens derived from the protein backbone.7–9 Evidence suggests that CO and methyl groups bind to Nip during catalysis.10, 11 Although aspects of the ACS catalytic mechanism remain uncertain, our understanding of it is gradually improving. The components of the A-cluster in its most oxidized redox state (called Aox) appear to be in the {[Fe4S4]2+ Nip2+ Nid2+} electronic configuration.12, 13 This state is inactive for both catalysis and methyl group transfer but it can be activated by a 2-electron reduction corresponding to an apparent midpoint potential of ca. −540 mV vs. NHE at neutral pH.14 The resulting reductively-activated state apparently has the cubane and Nid in the 2+ states, suggesting the unprecedented {[Fe4S4]2+ Nip0 Nid2+} configuration.12,13,14 Although the occurrence of a zero-valent Ni atom in the reductively activated state is not established, we will use this nomenclature throughout in this paper, for convenience if for no other reason. For a full discussion of this issue, readers are referred to the literature.4,13–15 If preferred, “Ni0” can be viewed simply as an electron-counting formalism indicating the Aox state to which 2e− have been added. This formal view does not complicate or bias any interpretation, analysis, or conclusion presented here. Whether the methyl group or CO bind first to the enzyme remains contentious, and reasonable arguments have been made for both cases.5,6,11,16 The one-electron-reduced and CO-bound state of the A-cluster (the S = ½ Ared-CO state) has been proposed to be an intermediate of catalysis as well as an inhibitory state. Recent evidence that ACS/CODH need not pass through the Ared-CO state during catalysis and evidence that reductive activation requires 2 electrons14 compel us to favor the case where the methyl group binds first. It is also known that the reductively activated state accepts a methyl group in the absence of CO, as shown in reaction 2. Ni0+CH3−Co3+FeSP⇄Ni2+−CH3+Co1+FeSPKmet=k+metk−met [2] The resulting methylated state is stable and has been characterized.11,14,17,18 When exposed to CO, e.g. during catalysis, CO is thought to insert into the Ni-methyl bond in accordance with reaction 3. Ni2+−CH3+CO⇄Ni2+−C(O)CH3Kins=k+insk−ins [3] Reaction of the acetyl intermediate with CoA affords acetyl-CoA and regenerates the reductively-activated state, reaction 4. Ni2+−C(O)CH3+CoA⇄CH3C(O)−CoA+Ni0KCoA=k+CoAk−CoA. [4] Reactions 2 – 4 complete the catalytic cycle for the synthesis of acetyl-CoA. Of these steps, only methyl group transfer, reaction 2, has been studied specifically.19–21 In a stopped-flow study, ACS/CODH and Ti3+citrate were preincubated to generate the Ni0 state, and then reacted against CH3-Co3+FeSP (also preincubated in Ti3+citrate) and monitored at 390 nm where the product Co1+FeSP absorbs. Under these conditions, the reverse of reaction 2 (i.e. starting from Ni2+-CH3 and Co1+) could barely be detected, indicating that the equilibrium position lies on the products side.21 In contrast, the reverse reaction proceeded rapidly and to near completion when the Ni2+-CH3 was not preincubated with Ti3+citrate. Under these conditions, the equilibrium position appears to be on the reactants side of reaction 2. This reductant-dependent shift in the kinetic and thermodynamic properties of the methyl group transfer reaction is not understood mechanistically. Bhaskar et al. have examined the steady-state kinetics of the exchange reaction between acetyl-CoA and dephospho-CoA as catalyzed by the ACS/CODH homolog from Methanosarcina barkeri.22 Their results indicate a ping-pong mechanism in which the binding of acetyl-CoA to the enzyme is followed by the release of CoA and formation of the acetyl-intermediate. They proposed that acetyl-CoA binds to the oxidized form of the enzyme, followed by reduction. This was suggested because partially-reduced enzyme exhibited cooperative binding with acetyl-CoA whereas fully reduced enzyme showed simple hyperbolic binding. However, the same behavior would be observed if enzyme were first reduced and then bound with acetyl-CoA. This latter scenario would be congruent with a nucleophilic attack (e.g. by a Ni0 species) on the carbonyl of acetyl-CoA, as in the reverse of reaction 4. The alternative proposal of binding followed by reduction would seem to require attack by Ni2+, a non-nucleophilic metal ion. Using two methods, Bhaskar et al. measured the equilibrium constant for the reverse of reaction 4 to be ~ 0.2 (averaged value),22 suggesting KCoA ~ 5 for a homologous ACS/CODH from a methanogenic archaeon. To date, no direct studies of the CO insertion reaction 3 have been reported, nor have the kinetics of the reductive elimination of the acetyl group and CoA, reaction 4, been reported. The problem in studying these reactions has been to identify strategies for monitoring them. Using stopped-flow kinetics, we report here that reactions 3 and 4 can be monitored by starting with the methylated state of ACS/CODH. Resulting traces were used to construct a simple kinetic model describing the catalytic mechanism of acetyl-CoA synthase. In this paper we report these results and describe the model.

Mössbauer and EPR spectroscopies were used to study the electronic structure of the A-cluster from recombinant acetyl-CoA synthase (the alpha subunit of the alpha2beta2 acetyl-CoA synthase/CO dehydrogenase). Once activated with Ni, these subunits have properties mimicking those associated with the alpha2beta2 tetramer, including structural heterogeneities. The Fe4S4 portion of the A-cluster in oxidized, methylated, and acetylated states was in the 2+ core oxidation state. Upon reduction with dithionite or Ti3+ citrate, samples of Ni-activated alpha developed the ability to accept a methyl group. Corresponding Mössbauer spectra exhibited two populations of A-clusters; roughly, 70% contained [Fe4S4]1+ cubanes, while approximately 30% contained [Fe4S4]2+ cubanes, suggesting an extremely low [Fe4S4](1+/2+) reduction potential for the 30% portion (perhaps-800 mV vs NHE). The same population ratio was observed when Ni-free unactivated alpha was used. The 70% fraction exhibited paramagnetic hyperfine structure in the absence of an applied magnetic field, excluding the possibility that it represents an [Fe4S4]1+ cluster coupled to a (proximal) Ni(p)1+. EPR spectra of dithionite-reduced, Ni-activated alpha exhibited features at g = 5.8 and g(ave) approximately 1.93, consistent with a physical mixture of {S = 3/2; S = 1/2} spin-states for A-clusters containing [Fe4S4]1+ clusters. Incubation of Ni-activated alpha with dithionite and CO converted 25% of alpha subunits into the S = 1/2 A(red)-CO state. Previous correlation of this state to functional A-clusters suggests that only the 30% fraction not reduced by dithionite or Ti3+ citrate represents functional A-clusters. Comparison of spin states in oxidized and methylated states suggests that two electrons are required for reductive activation, starting from the oxidized state containing Ni(p)2+. Refitting published activity-vs-potential data supports an n = 2 reductive activation. Enzyme starting in the methylated state exhibited catalytic activity in the absence of an external reductant, suggesting that the two electrons used in reductive activation are retained by the enzyme after each catalytic cycle and that the enzyme does not have to pass through the A(red)-CO state during catalysis. Taken together, our results suggest that a Ni(p)0 state may form upon reductive activation and reform after each catalytic cycle.

The default framework for modeling biochemical processes is that of a constant-volume reactor operating under steady-state conditions. This is satisfactory for many applications, but not for modeling growth and division of cells. In this study, a whole-cell modeling framework is developed that assumes expanding volumes and a cell-division cycle. A spherical newborn cell is designed to grow in volume during the growth phase of the cycle. After 80% of the cycle period, the cell begins to divide by constricting about its equator, ultimately affording two spherical cells with total volume equal to twice that of the original. The cell is partitioned into two regions or volumes, namely the cytoplasm (Vcyt) and membrane (Vmem), with molecular components present in each. Both volumes change during the cell cycle; Vcyt changes in response to osmotic pressure changes as nutrients enter the cell from the environment, while Vmem changes in response to this osmotic pressure effect such that membrane thickness remains invariant. The two volumes change at different rates; in most cases, this imposes periodic or oscillatory behavior on all components within the cell. Since the framework itself rather than a particular set of reactions and components is responsible for this behavior, it should be possible to model various biochemical processes within it, affording stable periodic solutions without requiring that the biochemical process itself generates oscillations as an inherent feature. Given that these processes naturally occur in growing and dividing cells, it is reasonable to conclude that the dynamics of component concentrations will be more realistic than when modeled within constant-volume and/or steady-state frameworks. This approach is illustrated using a symbolic whole cell model.

In eukaryotes, active transport involves motor proteins and cytoskeletal filaments. In contrast, bacteria (which lack cytoskeletal motor proteins) are thought to primarily rely on diffusion for molecular transport and cytoplasmic mixing. However, the physical nature of the bacterial cytoplasm remains poorly understood. Through single particle tracking of protein filaments, plasmids, storage granules and foreign particles of different sizes, we have found that the bacterial cytoplasm exhibits striking similarities to glass-forming liquids. Glass-forming liquids are noted for their metastability near the glass transition where their behavior changes from liquid-like to amorphous solid with even small perturbations. We find that particles of different sizes exhibit distinct dynamics and their mobility changes from fluid-like to glassy with increasing particle size. This size dependency provides an explanation for previous reports of both normal and anomalous diffusion in the bacterial cytoplasm. Moreover, we find that cellular metabolism attenuates the glassy properties of the bacterial cytoplasm. As a result, components that would otherwise be caged in narrow regions of confinement are able to explore the cytoplasmic space under metabolically active conditions. Cytoplasmic dynamics are directly impacted by environmental conditions that impact cellular metabolism. These findings have broad implications for our understanding of bacterial physiology as the glassy behavior of the cytoplasm impacts all intracellular processes involving large cellular components.

We have studied the kinetics of binding of dissolved antigens to antibody-receptors on the surface of the intact living cells. We have combined the Scanning Flow Cytometry with fluorescent immunoassay. Measurements of the kinetics were carried out for rabbit anti-mouse immunoglobulins that bind to receptors of the mouse hybridomas. Anti-mouse immunoglobulins were labeled with fluorescein isothiocyanate molecules (FITC). The individual cells were identified from the native Scanning Flow Cytometer records in our experiment. The kinetics of the formation of antigen-antibody complexes was measured from the fluorescence from the individual cells. A kinetic scheme for the heterogeneous reactions between ligands and receptors has been suggested. This scheme utilizes the concept of a distribution function of an amount of active receptors on the cell surface. The analytical solution for the system of the kinetic equations has been found in the reaction-limited approximation. The experimental kinetics were fitted by this mathematical model to determine the rate constant for binding reaction of the used antigen-antibody pair.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

SummaryCell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.